Project Summary The gut undergoes a profound ecological transition as the infant leaves the sterile (or near sterile) womb and acquires its microbiome. The microbiome encompasses not only the community of bacteria (bacterial microbiome), but also a diverse consortium of eukaryotic viruses and bacteriophages (virome). These early-in-life microbial communities are critical for healthy infant development including shaping growth, infection resistance, inflammation, and immune function. Alterations in these communities set the stage for neonatal adverse events, such as necrotizing enterocolitis, and later-in-life diseases such as obesity and allergies. Remarkably, the acquisition and early maturation of neonatal gut bacterial microbiome and virome are not well understood. Gut microbes are thought to be transmitted from the mother and the environment. We demonstrated a tremendous diversity of bacteriophages (viruses that infect bacteria) in stools from infants during their first 96 hours of life and then characterized a predator-prey dynamic between gut bacteriophages and bacteria in infants over the subsequent first two years of life. To determine if there is an in utero reservoir for these diverse bacteriophage communities, we characterized the virome and bacterial microbiota of amniotic fluid obtained at Cesarean section from 24 uncomplicated term pregnancies. Bacterial sequences in amniotic fluid were indistinguishable from those in buffer-alone contamination controls, viral reads were sparse in the amniotic fluid, and we found no evidence of a core viral community across samples. These data strongly suggest that the amniotic fluid does not give rise to the founding bacterial microbiome or virome. Multiple lines of evidence suggest that the environment is an important influence in shaping the bacterial microbiome and virome: gut microbial communities differ in content with geography, host genetics account for only a small fraction of the variability seen in bacterial microbiomes, and infants? bacterial microbiomes and viromes have little overlap with those of their mothers. However, we do not know the environmental source of most microbes that colonize the term infant. We are now compelled to conduct a systematic examination of the environment and determine the contribution of these ex utero exposures on colonization of the neonate. I propose to assemble a birth cohort and test my hypothesis that a portion of the infant gut virome and bacterial microbiome are acquired from the environment. Because of the enduring consequences of these founding microbial taxa for the infant, it is critical to understand how their acquisition occurs so that appropriate recommendations and interventions can be made to optimize this important event.